WO2024027793A1

WO2024027793A1 - Bispecific antibodies targeting ifnar1 and blys

Info

Publication number: WO2024027793A1
Application number: PCT/CN2023/111000
Authority: WO
Inventors: Jian Ding; Yong Zhao; Lei TENG; Dong DING; Yajuan WANG; Yunfei Zhang
Original assignee: I Mab Biopharma Hangzhou Co Ltd
Current assignee: I Mab Biopharma Hangzhou Co Ltd
Priority date: 2022-08-05
Filing date: 2023-08-03
Publication date: 2024-02-08
Anticipated expiration: 2025-02-05

Abstract

Provided are improved antibodies targeting the Blys protein as well as multi-specific antibodies that bind both IFNAR1 and Blys. These antibodies exhibited superior in vitro and in vivo properties and can be suitably use for treating diseases such as autoimmune diseases.

Description

BISPECIFIC ANTIBODIES TARGETING IFNAR1 AND BLYS

BACKGROUND

An autoimmune disease is a condition arising from an abnormal immune response to a functioning body part. The cause is typically unknown. Some autoimmune diseases such as lupus run in families, and certain cases may be triggered by infections or other environmental factors. A common example is systemic lupus erythematosus (SLE) , also known simply as lupus, characterized by heterogeneous clinical manifestations and the presence of multiple cellular and molecular abnormalities in the immune system, including leukocyte activation and cytokine dysregulation. Common symptoms include painful and swollen joints, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, feeling tired, and a red rash which is most commonly on the face. Often there are periods of illness, called flares, and periods of remission during which there are few symptoms.

The cause of SLE is not clear. The mechanism involves an immune response by autoantibodies against a person’s own tissues. These are most commonly anti-nuclear antibodies and they result in inflammation. There is no cure for SLE. SLE significantly increases the risk of cardiovascular disease with this being the most common cause of death.

Nephritis is an inflammation of the kidneys. The two most common causes of nephritis are infection and auto-immune processes. Nephritis can be a symptom of underlying conditions such as systemic lupus erythematosus (SLE) , diabetes, renal tuberculosis, or yellow fever. Glomerulonephritis is the most common type of nephritis and can include nephritic syndrome, nephrotic syndrome, and/or asymptomatic proteinuria and hematuria syndrome, all of which may lead to end stage renal disease (ESRD) and kidney failure. Lupus nephritis can involve various internal structures of the kidney and can include interstitial nephritis, glomerulonephritis (GN) , mesangial GN, membranous GN, diffuse proliferative GN and/or membranoproliferative GN. Similar to that of SLE, there is no definitive treatment or cure for LN. Aside from the foregoing mentioned cardiovascular risk, lupus nephritis (LN) is one of the most serious complications caused by SLE and remains a major cause of morbidity and mortality in SLE patients.

Treatments for SLE/LN may include NSAIDs, corticosteroids, immunosuppressants, hydroxychloroquine, and methotrexate. While many patients fail to respond or respond only partially to the above-mentioned standard of care medications, the long-term use of high doses of corticosteroids and cytotoxic therapies may have profound side effects such as bone marrow depression, increased infections with opportunistic organisms, irreversible ovarian failure, alopecia and increased risk of malignancy. Infectious complications coincident with active SLE and its treatment with immunosuppressive medications are the most common cause of death in patients with SLE. There is, therefore, a need for alternative therapies with better risk-benefit profiles for the treatment of SLE and/or lupus nephritis.

Type I interferons (IFN) and particularly IFNα, may play an important role in SLE pathogenesis. Both IFNα serum levels and expression of IFNα-inducible genes are consistently increased in SLE patients and usually correlate with disease activity and clinical manifestations. Binding of IFNα to the two-chain type I interferon receptor (IFNAR) initiates a signal transduction pathways that results in the expression of IFN-induced genes, most of them with immunoregulatory functions on B, T and NK lymphocytes, monocytes, macrophages, DCs and neutrophils. Inhibition of IFNα signaling, therefore, has the potential to treat SLE or LN.

The contribution of B-lymphocyte stimulator (BLyS) to autoantibody production and SLE disease exacerbation has also been recognized. BLyS is produced as a membrane form as well as a soluble protein by B lymphocytes, monocytes, neutrophils and plasmacytoid or myeloid DCs. Clinical studies have confirmed both circulating and cell surface BLyS overexpression in SLE patients and its correlation with the disease activity.

SUMMARY

Through extensive design and testing, the instant disclosure provides multi-specific antibodies that have superior binding affinities to both IFNAR1 and Blys proteins, have potent activities and high stabilities, and desirable pharmacokinetic performance.

According to one embodiment of the present disclosure, provided is a multi-specific antibody comprising an anti-IFNAR1 unit having binding specificity to interferon-alpha/beta receptor alpha chain (IFNAR1) , and an anti-Blys unit having binding specificity to B-lymphocyte stimulator (Blys) , wherein the anti-IFNAR1 unit comprises two pairs of heavy chain variable region (VH) and light chain variable region (VL) fused to the N-terminus of an IgG constant region, and wherein the anti-Blys unit comprises two single chain fragments (scFv) fused to the C-terminus of the IgG constant region.

In some embodiments, the scFv each is fused to the IgG constant region through a peptide linker having a length of 10-50 amino acid residues. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 2 or 3.

In some embodiments, each scFv comprises a VH and a VL connected through a second peptide linker having a length of 10-50 amino acid residues. In some embodiments, the second peptide linker comprises the amino acid sequence of SEQ ID NO: 2 or 3.

In some embodiments, the IgG constant region comprises human IgG1 CH domains and kappa constant domains. In some embodiments, the IgG1 CH domains comprise at least a substitution selected from the group consisting of L234F, L235E and P331S, according to Kabat numbering.

In some embodiments, the IgG1 CH domains further comprises one or more substitutions to extend the half-life. In some embodiments, the one or more substitutions are selected from the group consisting of (1) M428L/N434S (LS) ; (2) H285D/T307Q/A378V (DQV) ; (3) L309D/Q311H/N434S (DHS) ; and (4) M252Y/S254T/T256E (YTE) , according to Kabat numbering.

In some embodiments, the VH of the anti-IFNAR1 unit comprises the VH CDR1-3 of SEQ ID NO: 4, and the VL of the anti-IFNAR1 unit comprises the VL CDR1-3 of SEQ ID NO: 5. In some embodiments, the VH of the anti-IFNAR1 unit comprises the amino acid sequence of SEQ ID NO: 4, and the VL of the anti-IFNAR1 unit comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the VH of the anti-Blys unit comprises the VH CDR1-3 of SEQ ID NO: 11, and the VL of the anti-Blys unit comprises the VL CDR1-3 of SEQ ID NO: 17. In some embodiments, the VH of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 11 or has at least 85%sequence identity to SEQ ID NO: 11.

In some embodiments, the VH of the anti-Blys unit comprises at least one of 5V, 19K, 68T, 76S, 79Y, and 108L, according to Kabat numbering. In some embodiments, the VH of the anti-Blys unit comprises (a) 5V, 19K, 68T, 76S, 79Y, and 108L, (b) 19K, 68T, 76S, and 108L, (c) 5V, 68T, 76S, and 79Y, or (d) 19K and 108L. In some embodiments, the VL of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 17 or has at least 85%sequence identity to SEQ ID NO: 17. In some embodiments, the VL of the anti-Blys unit comprises 21I, according to Kabat numbering. In some embodiments, the VH of the anti-Blys unit further comprises 44C and the VL of the anti-Blys unit further comprises 100C, according to Kabat numbering.

In some embodiments, the VH of the anti-Blys unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-16, and the VL of the anti-Blys unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-19.

In some embodiments, the VH of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 14 or 16, and the VL of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the multi-specific antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 26, 28, 33 or 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 21.

Also provided, in one embodiment, is an antibody or antigen-binding fragment thereof having specificity to Blys, comprising a VH and a VL, wherein the VH (a) comprises the VH CDR1-3 of SEQ ID NO: 11, (b) has at least 85%sequence to SEQ ID NO: 11, and (c) comprises at least one of 5V, 19K, 68T, 76S, 79Y, and 108L, according to Kabat numbering, and wherein the VL comprises the VL CDR1-3 of SEQ ID NO: 17.

In some embodiments, the VH comprises (a) 5V, 19K, 68T, 76S, 79Y, and 108L, (b) 19K, 68T, 76S, and 108L, (c) 5V, 68T, 76S, and 79Y, or (d) 19K and 108L. In some embodiments, the VH comprises 5V, 19K, 68T, 76S, 79Y, and 108L. In some embodiments, the VH comprises 19K, 68T, 76S, and 108L.

In some embodiments, the VL has at least 85%sequence identity to SEQ ID NO: 17. In some embodiments, the VL comprises 21I, according to Kabat numbering.

In some embodiments, the VH comprises 44C and the VL comprises 100C, according to Kabat numbering. In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-16, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-19. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 14 or 16, and the VL comprises the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the multi-specific antibody or the antibody or antigen-binding fragment thereof is conjugated.

Also provided, in one embodiment, is a method of suppressing an immune response or treating an autoimmune disease or disorder in a patient in need thereof, comprising administering to the patient the multi-specific antibody or the antibody or antigen-binding fragment thereof of the present disclosure. Also provided is use of the multi-specific antibody or the antibody or antigen-binding fragment thereof for the preparation of a medicament for suppressing an immune response or treating an autoimmune disease or disorder in a patient in need thereof.

In some embodiments, the autoimmune disease or disorder is selected from the group consisting of type 1 diabetes, rheumatoid arthritis (RA) , psoriasis/psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (lupus) , lupus nephritis, inflammatory bowel disease, Addison’s disease, Graves’ disease, syndrome, Hashimoto’s thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and celiac disease. In some embodiments, the autoimmune disease or disorder is systemic lupus erythematosus or lupus nephritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a structure of certain bispecific antibodies tested in the examples.

FIG. 2A-B show that the tested bispecific antibodies have high binding affinity to the human BAFF (A) and IFNAR1 (B) proteins.

FIG. 3A-B show that the tested bispecific antibodies can potently induce proliferation of mouse splenocytes (A) and human primary B cells (B) .

FIG. 4A-C demonstrate the anti-IFNAR1 activities of the tested bispecific antibodies, through Daudi cell proliferation (A) , in an IFN-responsive reporter assay (B) , and in a dendritic cell development assay (C) .

FIG. 5A-5B show that the bispecific antibodies retained binding to FcRn, but did not bind to C1q. The anti-IFNAR1 antibody 8G11 was also named as TJ020811.

FIG. 6A-B show stability testing results of the bispecific antibodies.

FIG. 7A-F show dose-response (relative quantification and %of inhibition) curves for total B cell (CD19⁺) numbers (A-B) , CD69 MFI in CD19⁺ cells (C-D) , and plasma cell numbers (E-F) by Belimumab, Anifrolumab, their combo, and BsAbs.

FIG. 8A-B show the binding affinity of the Fc mutated L24 molecules to BAFF and IFNAR1 via ELISA.

FIG. 9A-B show the binding affinity of the Fc mutated L24 molecules to FcRn.

FIG. 10 shows the in vivo pharmacokinetics of the blood concentration of L24 (FES) and L24 (FES/LS) .

DETAILED DESCRIPTION

Definitions

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies. As such, the terms “a” (or “an” ) , “one or more, ” and “at least one” can be used interchangeably herein.

As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

The terms “antibody fragment” or “antigen-binding fragment” , as used herein, is a portion of an antibody such as F (ab’) ₂, F (ab) ₂, Fab’, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (V_H) and light chains (V_L) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V_H with the C-terminus of the V_L, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.

The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ l-γ4) . It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgG₅, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F (ab’) ₂, Fd, Fvs, single-chain Fvs (scFv) , single-chain antibodies, disulfide-linked Fvs (sdFv) , fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein) . Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda (K, λ) . Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining regions (CDRs) , of an antibody combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) . In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363: 446-448 (1993) .

In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest, ” Kabat, E., et al., U.S. Department of Health and Human Services, (1983) ; and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987) ) .

In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” ( “CDR” ) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. MoI. Biol. 196: 901-917 (1987) , which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

Antibodies disclosed herein may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be condricthoid in origin (e.g., from sharks) .

As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain) . As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG_l molecule and a hinge region derived from an IgG₃ molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgG_l molecule and, in part, from an IgG₃ molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG_l molecule and, in part, from an IgG₄ molecule.

As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) . The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161: 4083 (1998) ) .

As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system) .

As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In certain embodiments the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.

By “specifically binds” or “has specificity to, ” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B, ” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D. ”

As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.

Anti-IFNAR1/Blys Antibodies

Development of a successful bispecific antibody presents a significant challenge. The relevant strength and targeting epitopes of the two antibody units should be carefully balanced/coordinated, and efficacy and safety can often depend on the structure. Meanwhile, given that the structure of a bispecific antibody is more different from natural antibodies, its stability and manufacturability can also pose challenges.

The instant inventors designed and prepared a large number of bispecific antibodies targeting both the IFNAR1 (interferon-alpha/beta receptor alpha chain) and Blys (B-lymphocyte stimulator) proteins. The structure of these bispecific antibodies is illustrated in FIG. 1, which includes a full conventional Fab antibody targeting IFNAR1, and two identical single chain fragments (scFv) targeting Blys, each fused to the C-terminus of the two chains of the Fc fragment, through a peptide linker (linker 1) . The peptide linker between the VH and VL of the scFv is referred to as linker 2.

The tested bispecific antibodies are listed in Table 1A. All of the tested bispecific antibodies used the human IgG1/kappa isotype, while a few selected ones also had an “FES” version which annotates the IgG1 version with L234F/L235E/P331S (FES) substitutions in the Fc portion (all according to Kabat numbering) .

The anti-IFNAR1 VH/VL for all candidate bispecific antibodies were adopted from a humanized version of mouse antibody clone 8G11, 8G11-Hu. The VH sequence is provided in SEQ ID NO: 4 and the VL sequence is provided in SEQ ID NO: 5. The mouse VH/VL sequences (SEQ ID NO: 6/7) were used in bispecific antibody L23 and L23 (FES) as controls.

The VH/VL sequences of the anti-Blys scFv were adopted from anti-Blys antibody belimumab (Bel) . L5, L12, L13 and L23/L23 (FES) included the original VH and VL sequences (SEQ ID NO: 11 and 17, respectively) , while the remainder introduced various different mutations to the VH and VL sequences. All of them (L16-L22 and L24-L25, and their FES counterparts) introduced a G44C substitution to the VH and a G100C substitution to the VL, which could form a new inter-chain disulfide bond between the VH and the VL within the scFv.

L18, L20, L21, L22, L24 and L25 and their FES counterparts also included various other substitutions. For instance, L18/L24 and their FES counterparts included VKTSYL (Q5V/R19K/A68T/G76S/S79Y/M108L, Kabat numbering) , L20/L25 and their FES counterparts included a subset of VKTSYL, namely KTSL (R19K/A68T/G76S/M108L) , L21 included a different subset, KL (R19K/M108L) , and L22 included yet another subset, VTSY (Q5V/A68T/G76S/S79Y) . Meanwhile, L18, L20, L21, L24 and L25, and their FES counterparts also had a V21I substitution in their VL sequences.

Anti-Blys scFv, among these bispecific antibodies, adopted two VH/VL orientations. In L5, L24-L25 and their FES counterparts, the N-terminus to C-terminus orientation is VH-VL;in all the others, it is VL-VH.

Various sequences for linker 1 and linker 2 were also tested. For linker 1, the options were (G₄S) ₃ (SEQ ID NO: 1) and CTP (SEQ ID NO: 2) , which is the C-terminal peptide (CTP) of human chorionic gonadotropin (Hcg) beta subunit. For linker 2, the options were (G₄S) ₃ (SEQ ID NO: 1) , CTP (SEQ ID NO: 2) , and G₃-CTP-G₃ (SEQ ID NO: 3) .

As demonstrated in Example 2, L18, L20, L24 and L25 had higher binding affinity to Blys than the other tested bispecific antibodies. Take the comparison of L17 and L18, for example, the only structural difference between them is that L18 included VKTSYL in the VH and V21I in the VL (relative to the original belimumab sequences) . Likewise, the only difference between L19 and L20 is that 20 included KTSL in the VH and V21I in the VL. It can be concluded, therefore, that these substitutions (KTSL in VH and V21I in VL) improved the binding affinity of the anti-Blys unit. In Example 5, the receptor binding experiments confirmed that the FES substitutions are effective in improving the binding to FcRn and eliminating the binding to C1q.

Interestingly, in pharmacokinetic analysis of the bispecific antibodies (Example 8) , L24 (FES) and L25 (FES) showed the best performance. As these two differ from L18 (FES) and L20 (FES) only with respect to the VH/VL orientation, these data suggest that the VH-to- VL orientation in L24 (FES) and L25 (FES) helps to improve the pharmacokinetics of the bispecific antibodies.

With respect to the linker sequences, the data show that CTP (SEQ ID NO: 2) exhibited excellent performance as linker 1 overall, and both CTP (SEQ ID NO: 2) and G₃-CTP-G₃ (SEQ ID NO: 3) performed well as linker 2.

In accordance with one embodiment of the present disclosure, therefore, provided is a bispecific antibody, or a multi-specific antibody that incorporates the bispecific one, which includes an anti-IFNAR1 portion and an anti-Blys portion. In some embodiments, the bispecific antibody is dual valent to both IFNAR1 and Blys.

In some embodiments, the anti-IFNAR1 portion includes a conventional Fab antibody that includes two VH/VL pairs connected to IgG constant regions. In some embodiments, the IgG constant regions are human IgG1, IgG2, or IgG4 constant regions. In some embodiments, the IgG constant regions are human IgG1 constant regions, such as human IgG1 CH1, human IgG1 CH2, and human IgG1 CH3. In a particular embodiment, the IgG constant region includes human kappa chain (s) .

In some embodiments, the constant regions include substitutions that would alter the receptor binding properties. Example substitutions include one or more of L234F, L235E and P331S in the Fc region (Kabat numbering) . In some embodiments, the IgG constant region includes at least one of L234F, L235E and P331S. In some embodiments, the IgG constant region includes at least one of L234F and L235E, L234F and P331S, or L235E and P331S. In some embodiments, the IgG constant region includes all of L234F, L235E and P331S.

Further IgG1 Fc mutations include those that can enhance the pharmacokinetics of the bispecific antibodies, for example, half-life (T_1/2) , including but not limited to (1) M428L/N434S (LS or Xtend^TM) ; (2) H285D/T307Q/A378V (DQV) ; (3) L309D/Q311H/N434S (DHS) ; (4) M252Y/S254T/T256E (YTE) , (5) T250Q/M428L (QL) , (6) T307A/E380A/N434A (AAA) , and (7) V308P, according to Kabat numbering. More details can found in WO02060919, WO2004035752, WO2009086320, US20100104564, WO2018052556, WO2019033087, which are incorporated by reference in their entireties.

In some embodiments, the anti-Blys portion includes at least one, preferably two, single chain fragments (scFv) having binding specificity to Blys. In some embodiments, the scFv each is fused to the C-terminus of each of the constant regions (e.g., Fc) of the anti-IFNAR1 portion, optionally through a peptide linker.

In some embodiments, the peptide linker between the Fc and the scFv has as length that is from 0 to 60 residues. In some embodiments, the length of the peptide linker is 10-50 residues, from 12-45 residues, from 15-40 residues, or from 20 to 35 residues, without limitation. In one embodiment, the peptide linker includes or consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peptide linker includes or consists of the amino acid sequence of SEQ ID NO: 2. In one embodiment, the peptide linker includes or consists of the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the VH and the VL of the scFv are connected through a second peptide linker. In some embodiments, the second peptide linker between the VH and the VL in the scFv has as length that is from 0 to 60 residues. In some embodiments, the length of the second peptide linker is 10-50 residues, from 12-45 residues, from 15-40 residues, or from 20 to 35 residues, without limitation. In one embodiment, the second peptide linker includes or consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the second peptide linker includes or consists of the amino acid sequence of SEQ ID NO: 2. In one embodiment, the second peptide linker includes or consists of the amino acid sequence of SEQ ID NO: 3.

The VH and VL sequences in the scFv can take either orientation. When a linker is present between them, for instance, from the N-terminus to the C-terminus, the orientation can be VH-linker-VL, or VL-linker-VH. In a particular embodiment, the orientation can be VH-linker-VL, such as VH-SEQ ID NO: 2 or 3-VL.

Antibodies Suitable for Incorporation into Bispecific Antibodies

It is appreciated that the various disclosed embodiments, e.g., linker sequences, antibody structures, are applicable to any anti-IFNAR1 and anti-Blys antibodies and their antigen-binding fragments. Representative antibody sequences have also been tested and disclosed herein.

In one embodiment, the anti-IFNAR1 VH and VL sequences can be derived from murine antibody clone 8G11 or its humanized versions. In some embodiment, the VH includes CDR1, CDR2 and CDR3 of SEQ ID NO: 4 or 6 (as highlighted in Table 1B) . In some embodiment, the VL includes CDR1, CDR2 and CDR3 of SEQ ID NO: 5 or 7 (as highlighted in Table 1B) . In some embodiments, the VH includes the sequence of SEQ ID NO: 4 or 6, and the VL includes the sequence of SEQ ID NO: 5 or 7. In some embodiments, the VH and VL include the sequences of SEQ ID NO: 4 and 5, respectively.

In one embodiment, the anti-Blys VH and VL sequences can be derived from antibody belimumab (VH: SEQ ID NO: 11; VL: SEQ ID NO: 17) . In some embodiments, the belimumab VH/VL sequences can include further substitutions, which may be helpful in improving its properties. As demonstrated in the experimental examples, certain combinations of the Q5V, R19K, A68T, G76S, S79Y, and M108L (Kabat numbering) substitutions in the VH framework regions and/or the V21I in the VL framework region can improve the binding affinity.

In some embodiments, the present disclosure provides anti-Blys antibody, or antigen-binding fragment thereof, that includes a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH includes a VH CDR1, a VH CDR2, and a VH CDR3 of the VH CDR1-3 of SEQ ID NO: 11, and the VL includes a VL CDR1, a VL CDR2, and a VL CDR3 of the VL CDR1-3 of SEQ ID NO: 17.

In some embodiments, the VH includes at least one of 5V, 19K, 68T, 76S, 79Y, and 108L. In some embodiments, the VH includes at least two of 5V, 19K, 68T, 76S, 79Y, and 108L, such as 19K and 108L. In some embodiments, the VH includes at least three of 5V, 19K, 68T, 76S, 79Y, and 108L. In some embodiments, the VH includes at least four of 5V, 19K, 68T, 76S, 79Y, and 108L, such as 19K, 68T, 76S, and 108L, or 5V, 68T, 76S, and 79Y. In some embodiments, the VH includes all of 5V, 19K, 68T, 76S, 79Y, and 108L, in the framework region. In some embodiments, the VH further includes 44C.

In some embodiments, the VL includes 21I in the framework region. In some embodiments, the VL further includes 100C.

In some embodiments, the VH includes SEQ ID NO: 11. In some embodiments, the VH has at least 75%, 80%, 85%, 90%, 95%or 98%sequence identity to SEQ ID NO: 11, includes all of the CDRs of SEQ ID NO: 11 and includes one or more of the recited amino acids (e.g., 5V, 19K, 68T, 76S, 79Y, or 108L) in the framework region. In some embodiments, the VH has at least 75%, 80%, 85%, 90%, 95%or 98%sequence identity to SEQ ID NO: 11, includes all of the CDRs of SEQ ID NO: 11 and includes one or more of the recited amino acids (e.g., 5V, 19K, 68T, 76S, 79Y, 108L and 44C) in the framework region.

In some embodiments, the VL includes SEQ ID NO: 17. In some embodiments, the VL has at least 75%, 80%, 85%, 90%, 95%or 98%sequence identity to SEQ ID NO: 17, includes all of the CDRs of SEQ ID NO: 17 and includes 21I in the framework region. In some embodiments, the VL has at least 75%, 80%, 85%, 90%, 95%or 98%sequence identity to SEQ ID NO: 17, includes all of the CDRs of SEQ ID NO: 17 and includes 21I and 100C in the framework region.

Non-limiting examples of VH sequences include SEQ ID NO: 11-16. Non-limiting examples of VL sequences include SEQ ID NO: 17-19. In a particular example, the VH includes SEQ ID NO: 16 and the VL includes SEQ ID NO: 19. In a particular example, the VH includes SEQ ID NO: 14 and the VL includes SEQ ID NO: 19. In a particular example, the VH includes SEQ ID NO: 15 and the VL includes SEQ ID NO: 19. In a particular example, the VH includes SEQ ID NO: 13 and the VL includes SEQ ID NO: 19.

In a particular example, the VH includes SEQ ID NO: 16 and the VL includes SEQ ID NO: 18. In a particular example, the VH includes SEQ ID NO: 14 and the VL includes SEQ ID NO: 18. In a particular example, the VH includes SEQ ID NO: 15 and the VL includes SEQ ID NO: 18. In a particular example, the VH includes SEQ ID NO: 13 and the VL includes SEQ ID NO: 18.

Example Bispecific Antibody Sequences

Example bispecific antibodies incorporating certain features of the present technology are also provided. As demonstrated in the accompanying experimental examples, bispecific antibodies L18, L20, L24 and L25, as well as their FES counterparts had superior performances as compared to the other tested bispecific antibodies and benchmark antibodies.

Accordingly, one embodiment of the present disclosure provides a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 26, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 28, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 33, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 34, and two light chain each including the sequence of SEQ ID NO: 21.

In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 35, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 36, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 38, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 39, and two light chain each including the sequence of SEQ ID NO: 21.

In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 44, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 45, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 46, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 47, and two light chain each including the sequence of SEQ ID NO: 21.

In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 20, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 22, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 23, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 24, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 25, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 27, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 29, and two light chain each including the sequence of SEQ ID NO: 21. In one embodiment, provided is a bispecific antibody having two heavy chains each including the sequence of SEQ ID NO: 30, and two light chain each including the sequence of SEQ ID NO: 21.

It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to the starting sequence.

In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label) .

Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

In some embodiments, the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

The antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.

Polynucleotides Encoding the Antibodies and Methods of Preparing the Antibodies

The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties.

Treatments

As described herein, the antibodies (monospecific antibodies, bispecific antibodies, and multi-specific antibodies) , variants or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.

The present disclosure is further directed to antibody-based therapies which involve administering the antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein) .

One embodiment provides a method of suppressing an immune response in a patient in need thereof. The method entails administering to the patient an antibody, fragment, or bi-functional molecule of the present disclosure. In some embodiments, the patient is a tissue or organ transplant recipient.

In some embodiments, a method of treating an autoimmune disease or disorder is provided. Non-limiting examples of autoimmune disease or disorder include type 1 diabetes, rheumatoid arthritis (RA) , psoriasis/psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (lupus) , nephritis (e.g., interstitial nephritis, lupus nephritis) , inflammatory bowel disease, Addison’s disease, Graves’ disease, syndrome, Hashimoto’s thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and celiac disease.

In a particular embodiments, the method is useful for treating systemic lupus erythematosus (lupus) or lupus nephritis.

A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient’s age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

Methods of administration of the antibodies, variants or include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch) , bucally, or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

It may be desirable to administer the antibodies or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the disclosure, care must be taken to use materials to which the protein does not absorb.

In some embodiments, the antibodies of the disclosure are administered with a second therapeutic agent. The second therapeutic agent can be one or more immunosuppressive agents, non-steroidal anti-inflammatory drugs (NSAIDs) , disease modifying anti-rheumatic drugs (DMARDs) , hydroxychloroquine, methotrexate (MTX) , anti-B-cell surface marker antibodies, anti-CD20 antibodies, rituximab, TNF-inhibitors, corticosteroids, and co-stimulatory modifiers, or any combination thereof. Dosage regimes for the administration of the second therapeutic agent are well-known to a skilled person.

The second therapeutic agent as set forth herein are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99%of the heretofore-employed dosages. If such additional drugs are used at all, preferably, they are used in lower amounts than if the antibody or fragment were not present, especially in subsequent dosing beyond the initial dosing with the antibody or fragment of the disclosure, so as to eliminate or reduce side effects caused thereby.

The combined administration of a second therapeutic agent includes co-administration (concurrent administration) , using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) therapeutic agents (medicaments) simultaneously exert their biological activities.

Methods of detecting expression of a human interferon alpha and beta receptor subunit 1 (IFNAR1) protein in a sample are also provided, in some embodiments, comprising contacting the sample with the antibody or fragment thereof, and detecting the binding which indicates expression of IFNAR1 in the sample.

Compositions

The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody (monospecific antibody, bispecific antibody, and multi-specific antibody) , and an acceptable carrier. In some embodiments, the composition further includes a second therapeutic agent.

In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or Ph buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

EXAMPLES

Example 1. Generation of Anti-IFNAR-1/anti-Blys Antibodies

This example presents the design and sequences of anti-IFNAR1/anti-Blys antibodies.

All bispecific antibodies tested here employed a structure as illustrated in FIG. 1, which includes a conventional full-size Fab antibody at the N-terminal end targeting IFNAR1, and a single chain fragment (scFv) at the C-terminal end of the Fc fragment, targeting Blys. A “linker 1” links each of the two scFv to each CH3 fragment, and a “linker 2” is disposed between the VH and VL of the scFv. Table 1 provides the sequences and characteristics for each bispecific antibody.

Table 1A. Bispecific Antibodies

Notes:
Bel: Belimumab
H-L or L-H represents the order of VH and VL, from N-to C-terminus
VH G44C and VL G100C form a new inter-chain disulfide bond
FES: IgG1 constant region with L234F/L235E/P331S substitutions
LS: IgG1 constant region with M428L/N434S substitutions
DQV: IgG1 constant region with H285D/T307Q/A378V substitutions
DHS: IgG1 constant region with L309D/Q311H/N434S substitutions
YTE: IgG1 constant region with M252Y/S254T/T256E substitutions

Table 1B. Sequences

All of these tested bispecific antibodies included a scFv with VH/VL obtained from Belimumab ( “Bel” ) , or with VH/VL that contained further amino acid substitutions from Belimumab. In some of them, the VH/VL included a pair of VH G44C/VL G100C (Kabat numbering) substitutions (H44/L100) to introduce an interchain disulfide bond. Additional substitutions, in various combinations, included Q5V, R19K, A68T, G76S, S79Y, and M108L (Kabat numbering) . In the scFv, both VH-linker 2-VL and VL-linker 2-VH orientations were tested.

The anti-IFNAR-1 portion included VH/VL from a proprietary humanized antibody (8G11-Hu) , which was disclosed in PCT application WO2020057541A1, except L23 and L23 (FES) that used the chimeric version of 8G11 (8G11-Mu) and L23 was designated as Format 1 in WO2020057541A1.

Two versions of the constant regions were used. One of them is the conventional IgG1/kappa, and the other (referred to as “FES” ) included L234F/L235E/P331S (FES) substitutions in the Fc portion (Eu numbering) .

Two types of linker 1 sequences were tested, (G₄S) ₃ (SEQ ID NO: 1) and CTP (SSSSKAPPPSLPSPSRLPGPSDTPILPQ; SEQ ID NO: 2) . CTP is the C-terminal peptide (CTP) of human chorionic gonadotropin (Hcg) beta subunit. Linker 2 was (G₄S) ₃ (SEQ ID NO: 1) , CTP (SEQ ID NO: 2) , or G₃-CTP-G₃ (GGGSSSSKAPPPSLPSPSRLPGPSDTPILPQGGG; SEQ ID NO: 3) .

Example 2. Measurement of ELISA Binding/Blocking Activities

This example tested the activity of the bispecific antibodies at the molecular level in vitro, including binding and blocking experiments.

Methods

Binding of bispecific antibody to BAFF protein or IFNAR1 protein

A 96-well high affinity plate (Cat# 42592, CORNING) was coated with 1 μg/Ml human BAFF (Cat# C00D, Novoprotein) , murine BAFF (Cat# C06U, Novoprotein) and monkey BAFF (Cat# 90267-C01H, Sino biological) or human IFNAR1 (Cat# C358, Novoprotein) , murine IFNAR1 (Cat# 50469-M08H, Sino biological) and monkey IFNAR1 (Cat# 90113-C08H, Sino biological) protein solutions at 100 μl/well at 4℃, and was shaked overnight. The next day, the plate was washed 3 times with 300 μl PBST (Tween20: 0.5 ‰) , then blocked with 5%BSA/PBS at 100 μl/well for 1 hour, and shaked at room temperature. Washed the plate 3 times with 300 μl of PBST. Prepared the gradient dilution solution for the bispecific antibody sample in PBS. Added the solution to the 96-well plate at 100 μl/well and shaked at room temperature for 1 hour. Washed the plate for 3 times with 300 μl PBST. Prepared the secondary antibody (goat anti-human) IgG HRP (Cat# ab6858, Abcam) solution, added the solution to the 96-well plate at 100 μl/well, and shaked at room temperature for 30 min. Washed the plated for 4 times with 300 μl PBST. TMB (Cat# 34029, Thermo) was added at 100 μl/well for color development for 10 min and the reaction was terminated by adding 0.6N H₂SO₄ at 100 μl/well. The OD_450nm value was measured.

Blockade of binding of BAFF protein to its receptor by the bispecific antibody

A 96-well high affinity plate was coated with 1 μg/Ml human BAFF protein solution at 100 μl/well at 4℃, and was shaken overnight. The next day, the plate was washed 3 times with 300 μl of PBST (Tween20: 0.5 ‰) , then blocked with 5%BSA/PBS at 100 μl/well for 1 hour, and shaked at room temperature. Washed the plate 3 times with 300 μl of PBST. Prepared the gradient dilution solution for the antibody sample in PBS, added the solution to the 96-well plate at 100 μl/well, and shaken at room temperature for 1 hour. Washed the plate for 3 times with 300 μl of PBST. Prepared Biotin-labeled BAFF receptor solution [BCMA (Cat# 10620-H40H-B, Sino biological) at 0.2 μg/Ml or BR3 (Cat# 16079-H02H, Sino biological) at 0.8 μg/Ml or TACI (Cat# 29965-H02H, Sino biological) at 0.12 μg/Ml in PBS, added the solution to the 96-well plate at 100 μl/well, and shaked at room temperature for 1 hour. Washed the plate 3 times with 300 μl of PBST. Prepared Sreptavidin-HRP (Cat# A0303, Beyotime) solution, added the solution to the 96-well plate at 100 μl/well, and shaked at room temperature for 30 min. Washed the plate for 4 times with 300 μl of PBST. TMB was added at 100 μl/well for color development for 10 min and the reaction was terminated by adding 0.6N H₂SO₄ at 100 μl/well. The OD_450nm value was measured on a plate reader.

Results

The results are shown in FIG. 2A-B and Tables 2A-C. All of the tested bispecific antibodies exhibited excellent binding and blocking activities. In particular, L18, L20, L24 and L25 had the highest Blys-binding activities (Table 2A) . All of their FES counterparts exhibited excellent binding and blocking activities to human, mouse and Cyno Blys proteins, and to human and rhesus IFNAR1 proteins.

Table 2A. Binding to Human Blys

*Sample Binding EC50 (Nm) /Belimumab Binding EC50 (Nm) : antibody EC50 value is in front of “/” , and the positive
control Belimumab EC50 value in the same experiment is behind “/” .

Table 2B. Binding to Blys Protein of Different Species

Table 2C. Binding to IFNAR1 of Different Species

^# “-” represents no binding between the sample and Mouse IFNAR1.

Example 3. Cell-based Assays Assessing Anti-Blys Activity

Mouse splenocyte proliferation assay

This assay was conducted to validate the anti-Blys activity of the bispecific antibodies at the cellular level by observing the effect of the bispecific antibodies on the proliferation of mouse spleen cells.

Mouse spleen cells were collected and counted, centrifuged and re-suspended, and plated in a 96-well plate at 300,000 cells (in 70 μl of culture medium) per well. The 10x antibody sample gradient dilution working solution, the 10x LPS and 10xBlys protein working solutions were prepared in the culture medium. The blank well was added with 100 μl culture medium as Control; the Sample group was added with 70 μl cells + 10 μl antibody sample (10x) + 10 μl of LPS (10x) + 10 μl of BAFF (10x) ; the Control group was added with 70 μl of cells + 30 μl of culture medium; the LPS group was added with 70 μl of cells + 10 μl of LPS (10x) + 20 μl of culture medium; the LPS + Blys group was added with 70 μl of cells + 10 μl of LPS (10x) + 10 μl of culture medium + 10 μl of Blys (10x) . After incubation at 37℃ for 72 h, ATP-glo (Cat# G756, Promega) was added for cleavage, and the OD value was measured on a plate reader.

The testing results are shown in Table 3A and FIG. 3A. All of the tested bispecific antibodies exhibited potent anti-Blys activities.

Table 3A. Mouse splenocyte proliferation assay results

*Sample IC₅₀ (Nm) /Belimumab IC₅₀ (Nm) : the IC₅₀ value of the antibody sample is in front of “/” , and the IC₅₀
value of the positive control Belimumab in the same experiment is behind “/” .

Human primary B cell proliferation

This assay measure the anti-Blys activity of the bispecific antibody at the cellular level by observing the effect of the bispecific antibody molecule on the proliferation of human primary B cells.

Human CD19⁺ B cells were thawed, centrifuged, re-suspended, counted, and plated the cells in a 96-well plate at 1 × 10⁵ (in 35 Μl of culture medium) /well with two replicates. The complete culture medium is RPMI1640 + 10%FBS + 1%P/S+ 55 Μm mercaptoethanol. The 4x antibody sample Gradient dilution working solution, the 4x IL-4 (Cat# CX03, Novoprotein) and 4x Blys protein working solutions were prepared in the culture medium. The blank well was added with 140 μl of culture medium as Control; the Sample group was added with 35 μl cells + 35 μl antibody sample (4x) + 35 μl IL-4 (4x) + 35 μl Blys (4x) ; the Control group was added with 35 μl of cells + 105 μl culture medium; the IL-4 group was added with 35 μl cells + 35 μl IL-4 (4x) + 70 μl culture medium; the IL-4 + Blys group was added with 35 μl cells + 35 μl IL-4 (4x) + 35 μl culture medium + 35 μl Blys (4x) . After incubation at 37℃ for 6 days, ATP-glo was added for cleavage, and the OD value was measured on a plate reader.

As shown in FIG. 3B and Table 3B, all tested bispecific antibodies had potent anti-Blys activities. In fact, their activities were comparable to Belimumab.

Table 3B. Mouse splenocyte proliferation assay results

Example 4. Cell-based Assays Assessing Anti-IFNAR1 Activities

Daudi cell proliferation

This assay was conducted to measure the inhibitory activity of the bispecific antibody against type I interferon at the cellular level by observing the effect of the bispecific antibody molecule on the proliferation of Daudi cells.

The cultured Daudi cells were collected and counted, centrifuged and re-suspended, and plated in a 96-well plate at 1.8 × 10⁴ cells per well (in 80 μl of culture medium) . The 10x antibody sample gradient dilution working solution, and the 10x IFNa-2b (Cat# 13833-HNAY, Sino Biological) working solution were prepared in the culture medium. The blank well was added with 100 μl of medium as Control; the Sample group was added with 80 μl cells + 10 μl antibody sample (10x) + 10 μl IFNa-2b (10x) ; the Control group was added with 80 μl cells + 20 μl culture medium. For the Sample group, the antibody sample was added and incubated at 37℃ for 40 min before IFNa-2b was added. After incubation at 37℃ for 72 h, ATP-glo was added for cleavage, and the OD value was measured on a plate reader.

As shown in Table 4A and FIG. 4A, All the tested antibodies showed comparable activity as anifrolumab, in the rescue of IFN-induced inhibition of Daudi cells.

Table 4. Daudi cell proliferation results

*Sample IC₅₀ (μg/ml) /8G11 IC₅₀ (μg/ml) : the IC₅₀ value of the antibody sample is in front of “/” , and the IC₅₀
value of the positive control 8G11 monospecific antibody in the same experiment is behind “/” .

IFN-responsive reporter assay

This assay is to validate the IFNAR1-binding activity of the bispecific antibody at the cellular level.

The cultured HEK-Blue-IFNa Reporter cells were collected and counted, centrifuged and re-suspended the corresponding cells, and plateed in a 96-well plate at 5 × 10⁴ cells per well (in 80 μl of culture medium) . The 2x antibody sample gradient dilution working solution and the 10x IFNa-2b working solution were prepared in the culture medium. The blank well was added with 200 μl of medium as Control; the Sample group was added with 80 μl cells +100 μl antibody sample (10x) + 20 μl IFNa-2b (10x) ; to the Control group: add 80 μl cells +120 μl culture medium. In the Sample group, the antibody sample was added and incubated for 40 min before IFNa-2b was added. After incubation at 37℃ for 24 h, 20 μl of supernatant was transferred into the wells of a new 96-well plate, and each well was added and mixed with 180 μl prepared test solution. After incubation at 37℃ for 40 min, the OD_620nm value was measured on a microplate reader.

As shown in FIG. 4B and Table 4B, the tested antibodies showed comparable efficacy than anifrolumab in the IFN Reporter assay.

Table 4B. IFN-responsive reporter assay results

Dendritic Cell (DC) development assay

Monocytes differentiate into dendritic cells (DCs) when stimulated by GM-CSF and IFNa-2b, and their activity in blocking the binding of interferon to its receptor can be measured by adding the bispecific-antibody sample during the process.

The Monocytes isolated from human PBMCs were thawed, resuspended, counted and plated into a 24-well plate at 2 × 10⁵ cells per well (in 200 μl of culture medium) . The 5x antibody sample working solution, and the 5x IFNa-2b and 5x GM-CSF (Cat# CC79, Novoprotein) working solutions were prepared in the culture medium. The antibody sample, IFNa-2b and GM-CSF were added sequentially to corresponding wells at 100 μl per well, and incubated for 3 days. On the fourth day, the induced DC cells were collected well by well and counted sequentially. Then 2 × 10⁴ cells was removed from each cell sample, centrifuged with removal of the supernatant. The cells were washed twice with the complete culture medium, and resuspended to 100 μl for later use. Flow cytometry was performed on the remaining cells to determine CD86-APC expression. The CD4⁺ cells were thawed and counted, and mixed according to DC cell: CD4⁺ cell = 1: 5. The mixed cells were inoculated on a 96-well plate. After 5 days of co-incubation, the cell supernatant was taken to determine the level of IFN-γ.

As shown in FIG. 4C, the tested antibodies showed similar efficacy to anifrolumab in the DC development assay.

Example 5. Fc/C1q Binding abilities

This assay was conducted to measure the binding activity of bispecific antibody to FcRn.

The results of this experiment are obtained using the Novazzine ADD & Read FcRn binding kit (Cat# DD2408-00) . Detection principle: The kit contains Tag1-FcRn and two antibodies, including Anti-Tag1-Eu (fluorescent labeled donor Eu) and IgG-A2 (fluorescent labeled acceptor A2) . When FcRn interacts with IgG, Anti-Tag1-Eu and IgG-A2 get close to each other, and fluorescence resonance energy transfer (FRET) occurs. Eu is excited by 320 nm excitation light, and the donor emits 620 nm light to excite the fluorescence acceptor A2, and the acceptor emits 665 nm light. The FRET is disrupted by adding the test antibody to compete for FcRn binding. The concentration of the test sample added is inversely proportional to the FRET signal (665/620) .

The Anti-Tag1-Eu, Tag1-FcRn, and IgG-A2 working solutions were prepared. The 4x antibody sample gradient dilution working solution was prepared. The blank well was added with 5 μl Anti-Tag1-Eu + 5 μl IgG-A2 + 10 μl Diluent; the Sample group was added with 5 μl antibody sample + 5 μl anti-Tag1-FcRn + 5 μl Anti-Tag1-Eu + 5 μl IgG-A2; the Control group was added with 5 μl Diluent + 5 μl anti-Tag1-FcRn + 5 μl Anti-Tag1-Eu + 5 μl IgG-A2. After incubation at room temperature for 2 h, the excitation light at 320 nm was measured on a plate reader. The emission light at two wavelengths (665 nm and 620 nm) was measured.

As shown in Table 5A and FIG. 5A, all the tested antibodies showed higher FcRn binding activity than the benchmark antibody anifrolumab.

Table 5A. FcRn Binding Results

C1q Binding

This assay was conducted to measure the binding activity of the bispecific antibody to the C1q protein.

The test sample was diluted to 7 concentrations in 2-fold gradients starting from 150 Nm, and the plate was coated with 100 μl/well of the antibody of each concentration overnight at 4℃. The next day, washed the plate 3 times with 300 μl of PBST (Tween20: 0.5 ‰) , then blocked with 5%BSA/PBS at 100 μl/well for 1 hour, and shaked at room temperature. Washed the plate 3 times with 300 μl of PBST. The C1q working solution (2 μg/mL) was prepared in PBS, and added to a 96-well plate at 100 μl/well, and shaked at room temperature for 2 hours. The plate was washed 3 times with 300 μl of PBST. The Sreptavidin-HRP solution was prepared and added to the 96-well plate at 100 μl/well, and shaked at room temperature for 30 min. Washed the plate 4 times with 300 μl of PBST. TMB was added at 100 μl/well for color development for 10 min and the reaction was terminated by adding 0.6N H₂SO₄ at 100 μl/well. The OD_450nm value was measured on a plate reader.

The results are shown in Table 5B and FIG. 5B. None of the FES versions of the bispecific antibodies were able to bind to C1q.

Table 5B. C1q binding activity

* “-” indicates that the sample is not bound to C1q.

Example 6. Antigen-Antibody Affinity Tests

Binding Affinity to Blys

The real-time monitoring of the molecular binding process was performed to determine the binding constant (k_a or k_on) and dissociation constant (k_d or k_off) , as well as the rate of initial binding. The antibody to the antigen binding affinity (K_D) was obtained by fitting calculation and analysis.

The affinity of each antibody to human Blys was detected by the ForteBio technique. An appropriate amount of the antigen was diluted to 20 μg/mL. The diluted antigen was used as loading material and stored at 4℃ for later use. An appropriate amount of the antibody was diluted to 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0 Nm sequentially (the specific concentration points used for experiment analysis is provided in the results) . The diluted antibody samples are used as the samples (analytes) and stored at 4℃ for later use. The blank solution is recorded as 0 Nm as the control (analyte) , and 25 nM is recorded as the non-specific binding site (analyte) .

As shown in Table 6A, all of the tested antibodies had strong affinity to the Blys protein.

Table 6A. Blys-binding affinities

Binding Affinity to IFNAR1

Real-time monitoring of the molecular binding is performed to determine the binding constant (k_a or k_on) and dissociation constant (k_d or k_off) , as well as the rate of initial binding. The antibody to the antigen binding affinity (K_D) was obtained by fitting calculation and analysis.

Binding affinity of each antibody to human IFNAR1 is detected by Biacore technique. The antibody solution at 2 μg/mL or 4 μg/mL was incubated with protein A chip for 30 s for antibody capture. During the antigen binding phase, human IFNAR1 protein as the mobile phase binds to the antibody captured on the sensor chip for 120 s. During the dissociation phase, the antibody-antigen complex was eluted with HBS-EP buffer for 360 s. Antigen binding to antibody on the sensor chip is quantified by Biacore 8K.

As shown in Table 6A, all of the tested antibodies had strong affinity to the human IFNAR1 protein.

Table 6B. IFNAR1-binding affinities

Example 7. Antibody Stability

Expression Level, Purity, Thermal Stability Tm

Different molecular expression properties, including expression level and purity, and thermal stability were measured in this assay. The results are shown in Table 7A.

Table 7A. Stability testing results

* Thermostability Tm of two molecules, L5 and L13, was not tested.

Accelerated Stability at 37℃

To test accelerated stability, the antibodies were placed at 37℃ for a period of time to accelerate the denaturation process.

Four ampules of each antibody was placed in an incubator at 37℃ for 0, 1, 2 and 3 weeks, respectively, and collected for subsequent testing. The antibody stability was validated by ELISA Binding test. The results are shown in Table 7B.

As shown in Tables 7A-B, these tested antibodies exhibited excellent stabilities.

Table 7B. Accelerated stability testing results

Example 8. In Vivo PK

This example checked the metabolism of the tested antibody molecules in mice.

Balb/c mice (n = 3) were injected intravenously with an antibody solution at a dose of 5 mg/kg. Orbital blood was collected at 0, 4, 7, 24, 72, 120, 168, 240, 288, 336, 384, 456 and 504 hours before and after dosing. The plasma concentrations was measured by ELISA, and the concentration-time curve was plotted.

1 μg/mL Human IFNAR1 or Blys was coated on the plate overnight, and washed with 300 μl of PBST on the next day. Each well was blocked with 100 μl 5%BSA for 1 h at room temperature. The standard curve was prepared using the remaining antibody samples (0.5 mg/mL) from the animal experiment. 2.4 μl antibody solution was diluted 166.7 fold with 400 μl PBS to 3 μg/mL, and 200 μl antibody solution was diluted with 200 μl of PBS, and so forth. 12 concentrations were obtained by 2-fold gradients, and each well was added with 1%blank balb/c serum. Serum sample was prepared by adding 1 μl antibody of 4, 7, 24 and 72 h respectively into 19 μl blank C57 serum for 20-fold dilution, then 2 μl of the diluted solution was added into 198 μl PBS for 100-fold dilution; 2 μl antibody solution of the 120 and 168 h respectively was added into 18 μl blank C57 serum for 10-fold dilution, then 2 μl diluted solution was added into 198 μl PBS for 100-fold dilution; 2 μl antibody solution of the 0, 240, 288, 336, 384, 456 and 504 h respectively was added into 198 μl of PBS for 100-fold dilution. The plate was washed 3 times with 300 μl PBST, and 100 μl prepared antibody solution was added to each well, and incubated at room temperature for 1 h. The plate was washed 3 times with 300 μl PBST, and 100 μl goat-anti-human IgG bispecific antibody was added to each well and incubated at room temperature for 1 h. The plate was washed 3 times with 300 μl of PBST. TMB was added for color development for 5 min and the reaction was terminated by adding 0.6N H₂SO₄ at 100 μl/well. The OD_450nm value was measured.

The results are shown in FIG. 6A-B. Among all tested antibodies, L24 (FES) and L25 (FES) showed the best pharmacokinetic performance.

Example 9. In Vitro inhibition of B cell activation and plasma cell differentiation

It was reported previously that in a B cell and plasmacytoid dendritic cell (pDC) co-culture system, the TLR9 agonist CpG-A oligonucleotide (ODN) 2216 promoted the differentiation of B cells into plasma cells in a pDC-dependent manner. 1 CpG-Astimulated pDCs to produce IFN-α. Anifrolumab, an anti-interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody inhibited plasma cell differentiation stimulated by CpG-A in the B cell/pDC co-cultures.

The aim of the present study was to evaluate the efficacy of two bispecific antibodies (BsAbs) , L24 (FES) and L25 (FES) , directed against both B-cell activating factor (BAFF) and interferon (IFN) alpha receptor 1 (IFNAR1) in the inhibition of B cell activation (CD69 induction, total B cell number) and plasma cell differentiation stimulated by CpG-A in a B cell and plasmacytoid dendritic cell (Pdc) co-culture system.

B cells (CD19⁺, 1x10⁵) and Pdc (Lin^-/HLA-DR⁺/CD123⁺, 1x10⁴) were co-cultured and treated with 0.5 μM CpG-A for 6 days. B cell activation was assessed by CD69 expression levels and the number of CD19⁺/CD27^hi/CD38^hi plasma cells was determined, both using flow cytometry. The ability of anti-BAFF monoclonal antibody Belimumab, anti-IFNAR1 monoclonal antibody Anifrolumab, and the BsAbs L24 (FES) and L25 (FES) to inhibit B cell activation and plasma cell differentiation was evaluated.

CpG-A promoted B cell activation and plasma cell differentiation in a Pdc-dependent manner. As shown in FIG. 7A-F, Belimumab had a minimal to moderate effect while Anifrolumab potently inhibits B cell activation and plasma cell differentiation, indicating that type I IFN plays a more important role than BAFF in this system. The BsAbs L24 (FES) and L25 (FES) showed a better efficacy than Belimumab and Anifrolumab in dose-response experiments. L24 (FES) also showed comparable efficacy as the Belimumab/Anifrolumab combo treatment in dose-response experiment.

Example 10. Enhanced in vitro efficacy of the Fc mutations for the BsAbs

To enhance the antibody’s pharmacokinetics by extending the antibody’s half-life in the body, four L24 variants with Fc mutations were prepared and tested: (1) M428L/N434S (L24 (FES/LS) ) ; (2) H285D/T307Q/A378V (L24 (FES/DQV) ) ; (3) L309D/Q311H/N434S (L24 (FES/DHS) ) ; and (4) M252Y/S254T/T256E (L24 (FES/YTE) ) , see Table 1A and 1B.

Expression, purity and thermostability Tm

The antibody expression, purity and thermostability of the mutated L24 molecules are shown in Table 8.

Table 8. L24 Expression, purity and thermostability

Binding affinity

Binding affinity of the two arms (anti-BAFF and anti-IFNAR1) on the Fc mutated L24 was tested via ELISA.

96-well high-affinity plates (Cat# 42592, CORNING) were coated with 100μL/well of either human BAFF protein solution (Cat# C00D, Novoprotein) at a concentration of 1μg/μl or human IFNAR1 protein solution (Cat# C358, Novoprotein) and incubated at 4℃with shaking overnight. The next day, washed the plates three times with 300Μl PBST (0.5‰Tween20) each time. The plates were then blocked with 100μl/well of 5%BSA/PBS and shaked at room temperature for 1 hour. The plates were washed three times with 300μl PBST each time. A gradient dilution solution of the dual antibody samples was prepared using PBS. 100Μl/well of the dilution solution was added to the 96-well plate and shaked the plates at room temperature for 1 hour. Washed the plates three times with 300μl PBST each time. The secondary antibody goat anti-human IgG HRP solution (Cat# ab6858, Abcam) were prepared and was added 100μl/well to the 96-well plate. Shaked the plates at room temperature for 30 minutes. Washed the plates four times with 300μl PBST each time. 100μl/well of TMB (Cat# 34029, Thermo) was added and the plates were incubated for 10 minutes for color development. Finally, 100μl/well of 0.6N H₂SO₄ was added to stop the color development and the OD450 nm was measured.

Results are shown in Table 9 and FIG. 8A-B. Both arms exhibit similar binding affinity with original L24 or L24 (FES) molecules.

Table 9. Binding affinity of L24 Fc mutations.

FcRn Binding

Competition Kit: The experiment results were tested using the Novex ADD&Read FcRn Binding Kit (Cat# DD2408-00) . The detection principle of the kit involves Tag1-FcRn and two antibodies: Anti-Tag1-Eu (labeled with fluorescence donor Eu) and IgG-A2 (labeled with fluorescence acceptor A2) . When FcRn interacts with IgG, Anti-Tag1-Eu and IgG-A2 come close together, leading to fluorescence resonance energy transfer (FRET) . Excitation light at 320nm stimulates Eu, and the donor emits light at 620nm, exciting the acceptor A2 to emit light at 665nm. When the test antibody sample is added, it competes for binding with FcRn, disrupting FRET. The concentration of the test sample is inversely proportional to the FRET signal (665/620) .

To perform the experiment, Anti-Tag1-Eu, Tag1-FcRn, and IgG-A2 working solutions were prepared (the stock solution is 25x concentrated) . A gradient dilution working solution of the 4x antibody sample was also prepared. The blank wells were added with 5μl Anti-Tag1-Eu + 5μl IgG-A2 + 10Μl Diluent. The sample wells were added with 5μl antibody sample + 5μl anti-Tag1-FcRn + 5μl Anti-Tag1-Eu + 5μl IgG-A2. The control wells were added with 5μl Diluent + 5μl anti-Tag1-FcRn + 5μl Anti-Tag1-Eu + 5μl IgG-A2. The plates were incubated at room temperature for 2 hours, followed by detection using an enzyme- linked immunosorbent assay (ELISA) reader with excitation light at 320nm and measuring the emission at two wavelengths (665nm and 620nm) .

In another part of the experiment (ELISA Binding at pH 7.4) , human FcRn protein solution at a concentration of 1μg/ml was coated onto a 96-well high-binding plate (Cat# 42592, CORNING) at 100μl/well, and left to incubate overnight at 4℃ with gentle shaking. The following day, the plate was washed three times with 300 μL PBST (Tween20: 0.5‰; pH 7.4) , and then blocked with 5%BSA/PBS at 100μl/well for 1 hour at room temperature with shaking. After washing the plate three times with 300 μL PBST (pH 7.4) , a gradient dilution solution of the dual antibodies was prepared in PBS (pH 7.4) and added to the plate at 100μl/well for 1 hour with shaking at room temperature. The plate was washed again with 300 μL PBST (pH 7.4) three times. Next, a solution of secondary goat anti-human IgG HRP (Cat# ab6858, Abcam) at 100μl/well was added to the plate and incubated for 1 hour at room temperature with shaking. The plate was washed three times with PBST (pH 7.4) . Then, 100μl of TMB substrate (Cat# 34029, Thermo) was added to each well for 5 minutes of color development. Finally, 100 μl of 0.6N H₂SO₄ was added to each well to stop the color development, and the optical density at 450nm was measured.

Results are shown in Table 10 and FIG. 9A-B. All four Fc mutated L24 molecules showed significantly increased binding to the FcRn, while no binding was detected at Ph 7.4.

Table 10. FcRn binding of the four Fc mutated L24 molecules.

Example 11. Enhanced in vivo efficacy of the Fc mutations for the BsAbs

FcRn trangenic mice (n=4 in each group) were administered intravenously with the Fc mutated L24 (Y05-L24 (FES) , Y05-L24 (FES/LS) ) at 5mg/kg in PBS solution. Each group of mice will be subjected to blood collection before drug administration (0hr) and at 5min, 2hr, 4hr, 7hr, 24hr (1d) , 72hr (3d) , 120hr (5d) , 168hr (7d) , 240hr (10d) , 336hr (14d) , 408hr (17d) , 480hr (20d) after drug administration. The collected blood will be processed to obtain serum samples, which will be stored at -60℃ to -80℃. The serum samples will be analyzed for blood concentration using the established ELISA method.

Results are shown in FIG. 10. Under the conditions of this experiment, the blood concentration data shows that the pharmacokinetic properties of the L24 (FES/LS) molecule are significantly better than the L24 (FES) molecule in FcRn transgenic mice. The predicted half-life (T_1/2) are improved from 67.4 hours to 137 hours, and the exposure level (AUC0-t) is improved from 5590 hr*ug/mL to 13697 hr*ug/mL.

* * *

The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

A multi-specific antibody comprising an anti-IFNAR1 unit having binding specificity to interferon-alpha/beta receptor alpha chain (IFNAR1) , and an anti-Blys unit having binding specificity to B-lymphocyte stimulator (Blys) , wherein the anti-IFNAR1 unit comprises two pairs of heavy chain variable region (VH) and light chain variable region (VL) fused to the N-terminus of an IgG constant region, and wherein the anti-Blys unit comprises two single chain fragments (scFv) fused to the C-terminus of the IgG constant region.
The multi-specific antibody of claim 1, wherein the scFv each is fused to the IgG constant region through a peptide linker having a length of 10-50 amino acid residues.
The multi-specific antibody of claim 2, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 2 or 3.
The multi-specific antibody of any preceding claim, wherein each scFv comprises a VH and a VL connected through a second peptide linker having a length of 10-50 amino acid residues.
The multi-specific antibody of claim 4, wherein the second peptide linker comprises the amino acid sequence of SEQ ID NO: 2 or 3.
The multi-specific antibody of any preceding claim, wherein the IgG constant region comprises human IgG1 CH domains and kappa constant domains.
The multi-specific antibody of claim 6, wherein the IgG1 CH domains comprise at least a substitution selected from the group consisting of L234F, L235E and P331S, according to Kabat numbering.
The multi-specific antibody of claim 6 or 7, wherein the IgG1 CH domains further comprises one or more substitutions to extend the half-life.
The multi-specific antibody of claim 9, wherein the one or more substitutions are selected from the group consisting of (1) M428L/N434S (LS) ; (2) H285D/T307Q/A378V (DQV) ; (3) L309D/Q311H/N434S (DHS) ; and (4) M252Y/S254T/T256E (YTE) , according to Kabat numbering.
The multi-specific antibody of any preceding claim, wherein the VH of the anti-IFNAR1 unit comprises the VH CDR1-3 of SEQ ID NO: 4, and the VL of the anti-IFNAR1 unit comprises the VL CDR1-3 of SEQ ID NO: 5.
The multi-specific antibody of claim 10, wherein the VH of the anti-IFNAR1 unit comprises the amino acid sequence of SEQ ID NO: 4, and the VL of the anti-IFNAR1 unit comprises the amino acid sequence of SEQ ID NO: 5.
The multi-specific antibody of any preceding claim, wherein the VH of the anti-Blys unit comprises the VH CDR1-3 of SEQ ID NO: 11, and the VL of the anti-Blys unit comprises the VL CDR1-3 of SEQ ID NO: 17.
The multi-specific antibody of claim 12, wherein the VH of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 11 or has at least 85%sequence identity to SEQ ID NO: 11.
The multi-specific antibody of claim 13, wherein the VH of the anti-Blys unit comprises at least one of 5V, 19K, 68T, 76S, 79Y, and 108L, according to Kabat numbering.
The multi-specific antibody of claim 14, wherein the VH of the anti-Blys unit comprises (a) 5V, 19K, 68T, 76S, 79Y, and 108L, (b) 19K, 68T, 76S, and 108L, (c) 5V, 68T, 76S, and 79Y, or (d) 19K and 108L.
The multi-specific antibody of any one of claims 13-15, wherein the VL of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 17 or has at least 85%sequence identity to SEQ ID NO: 17.
The multi-specific antibody of claim 16, wherein the VL of the anti-Blys unit comprises 21I, according to Kabat numbering.
The multi-specific antibody of any one of claims 13-15, wherein the VH of the anti-Blys unit further comprises 44C and the VL of the anti-Blys unit further comprises 100C, according to Kabat numbering.
The multi-specific antibody of claim 12, wherein the VH of the anti-Blys unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-16, and the VL of the anti-Blys unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-19.
The multi-specific antibody of claim 12, wherein the VH of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 14 or 16, and the VL of the anti-Blys unit comprises the amino acid sequence of SEQ ID NO: 19.
The multi-specific antibody of claim 1, which comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 26, 28, 33 or 34, and two light chains each comprising the amino acid sequence of SEQ ID NO: 21.
An antibody or antigen-binding fragment thereof having specificity to Blys, comprising a VH and a VL, wherein the VH (a) comprises the VH CDR1-3 of SEQ ID NO: 11, (b) has at least 85%sequence to SEQ ID NO: 11, and (c) comprises at least one of 5V, 19K, 68T, 76S, 79Y, and 108L, according to Kabat numbering, and wherein the VL comprises the VL CDR1-3 of SEQ ID NO: 17.
The antibody or antigen-binding fragment thereof of claim 20, wherein the VH comprises (a) 5V, 19K, 68T, 76S, 79Y, and 108L, (b) 19K, 68T, 76S, and 108L, (c) 5V, 68T, 76S, and 79Y, or (d) 19K and 108L.
The antibody or antigen-binding fragment thereof of claim 22, wherein the VH comprises 5V, 19K, 68T, 76S, 79Y, and 108L.
The antibody or antigen-binding fragment thereof of claim 22, wherein the VH comprises 19K, 68T, 76S, and 108L.
The antibody or antigen-binding fragment thereof of any one of claims 22-25, wherein the VL has at least 85%sequence identity to SEQ ID NO: 17.
The antibody or antigen-binding fragment thereof of claim 26, wherein the VL comprises 21I, according to Kabat numbering.
The antibody or antigen-binding fragment thereof of any one of claims 22-27, wherein the VH comprises 44C and the VL comprises 100C, according to Kabat numbering.
The antibody or antigen-binding fragment thereof of claim 22, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-16, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-19.
The antibody or antigen-binding fragment thereof of claim 29, wherein the VH comprises the amino acid sequence of SEQ ID NO: 14 or 16, and the VL comprises the amino acid sequence of SEQ ID NO: 19.
The multi-specific antibody of any one of claims 1-21 or the antibody or antigen-binding fragment thereof of any one of claims 22-30, wherein the multi-specific antibody or the antibody or antigen-binding fragment thereof is conjugated.
One or more polynucleotide (s) encoding the multi-specific antibody of any one of claims 1-19, or the antibody or antigen-binding fragment thereof of any one of claims 22-30.
An isolated cell comprising the one or more polynucleotide (s) of claim 32.
A composition comprising the multi-specific antibody of any one of claims 1-21, or the antibody or antigen-binding fragment thereof of any one of claims 22-31 and a pharmaceutically acceptable carrier.
The composition of claim 34, comprising a second therapeutic agent.
A method of suppressing an immune response or treating an autoimmune disease or disorder in a patient in need thereof, comprising administering to the patient the multi-specific antibody of any one of claims 1-21, or the antibody or antigen-binding fragment thereof of any one of claims 22-31.
Use of the multi-specific antibody of any one of claims 1-21, or the antibody or antigen-binding fragment thereof of any one of claims 22-31 for the preparation of a medicament for suppressing an immune response or treating an autoimmune disease or disorder in a patient in need thereof.
The method of claim 34 or claim 35 or the use of claim 37, for treating an autoimmune disease or disorder.
The method of claim 34 or claim 35 or the use of claim 37, wherein the treatment further comprising administering to the patient with a second therapeutic agent.
The method of claim 34 or claim 35 or the use of claim 37, wherein the autoimmune disease or disorder is selected from the group consisting of type 1 diabetes, rheumatoid arthritis (RA) , psoriasis/psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (lupus) , lupus nephritis, inflammatory bowel disease, Addison’s disease, Graves’ disease, syndrome, Hashimoto’s thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and celiac disease.
The method of claim 34 or claim 35 or the use of claim 37, wherein the autoimmune disease or disorder is systemic lupus erythematosus or lupus nephritis.
A method of producing the multi-specific antibody of any one of claims 1-21, or the antibody or antigen-binding fragment thereof of any one of claims 22-31, comprising culturing the isolated cell of claim 33 under the condition at which the polynucleotide (s) of claim 32 is expressed.